The Role of Cation Binding in Determining Substrate Selectivity of Glutamate Transporters

Abstract

Glutamate transport is coupled to the co-transport of 3Na ؉ and 1H؉ and the countertransport of 1 K ؉ . However, the mechanism of how this process occurs is not well understood. The crystal structure of an archaeal homolog of the human glutamate transporters, Glt Ph , has provided the framework to begin to understand the mechanism of transport. The glutamate transporter EAAT2 is different from other subtypes in two respects. First, Li ؉ cannot support transport by EAAT2, whereas it can support transport by the other excitatory amino acid transporters, and second, EAAT2 is sensitive to a wider range of blockers than other subtypes. We have investigated the relationship between the cation driving transport and whether the glutamate analogues, L-anti-endo-3,4-methanopyrrolidinedicarboxylic acid (MPDC) and (2S,4R)-4-methylglutamate (4MG), are substrates or blockers of transport. We have also investigated the molecular basis for these differences. EAAT2 has a Ser residue at position 441 with hairpin loop 2, whereas the corresponding residue in EAAT1 is a Gly residue. We demonstrate that if the transporter has a Ser residue at this position, then 4MG and MPDC are poor substrates in Na ؉ , and Li ؉ cannot support transport of any substrate. Conversely, if the transporter has a Gly residue at this position, then in Na ؉ 4MG and MPDC are substrates with efficacy comparable with glutamate, but in Li ؉ 4MG and MPDC are poor substrates relative to glutamate. This Ser/Gly residue is located between the bound substrate and one of the cation binding sites, which provides an explanation for the coupling of substrate and cation binding.

Substrate-dependent Gating of Anion Channels Associated with Excitatory Amino Acid Transporter 4

“…Our data differ from recent reports that Li ϩ cannot support activation of EAAT1 and EAAT3 anion currents by glutamate (20,21). A possible explanation for this discrepancy might be that Li ϩ only activates EAAT anion channels in the presence of internal Na ϩ , however this is not the case.…”

“…Mutating this amino acid located at the tip of hairpin 2 (25,30) to glycine or performing the reverse mutation in the EAAT3 background permitted or prohibited Li ϩ to drive transport (28). In EAAT1 and EAAT3, Li ϩ was reported to support coupled transport but apparently not anion currents (20,21). We decided to reanalyze the effects of Li ϩ because these earlier results indicated cation-dependent anion channel gating beyond the uptake cycle.…”

“…However, defining kinetic parameters from anion currents is only possible if all channel states are directly associated with the uptake cycle. This has been questioned by recent reports: for EAAT1 and EAAT3, Li ϩ was reported to support coupled transport, but not anion channel function, indicating an additional Na ϩ -dependent conformational gating transition of EAAT anion channels beyond the uptake cycle (20,21).…”

“…However, defining kinetic parameters from anion currents is only possible if all channel states are directly associated with the uptake cycle. This has been questioned by recent reports: for EAAT1 and EAAT3, Li ϩ was reported to support coupled transport, but not anion channel function, indicating an additional Na ϩ -dependent conformational gating transition of EAAT anion channels beyond the uptake cycle (20,21).To further probe the existence of such conformational changes, we studied the substrate dependence of EAAT4 anion channels by comparing channel gating in Na ϩ and Li ϩ as well as in glutamate, aspartate, and cysteine. We observed distinct anion channel gating for the three amino acid substrates.…”

Loss-of-function mutations in the glutamate transporter SLC1A1 cause human dicarboxylic aminoaciduria

“…Radiolabeled glutamate and cysteine uptake and electrophysiological studies were performed in X. laevis oocytes after injection of in vitro-transcribed cRNA as previously described (34,42,43). Substrate uptake was measured by placing 5 oocytes in ND96 buffer containing 3 H-l-glutamate (GE Healthcare) or 35 S-l-cysteine (Perkin Elmer).…”